Cancer is one of the most enduring health problems of the modern era. Recent developments using immunological techniques offer substantial new promise for developing vaccines against immunogenic cancers. Over the past year, we have been developing a method for educating the endogenous immune system to recognize and target tumor cells. The approach capitalizes on the specialized functions of dendritic cells (DCs) to recognize antigens and induce differentiation of nave T cells into cytotoxic T cells to eliminate tumor cells. While this approach is highly promising, we believe that the development of DC-based cancer vaccines will be substantially enhanced by exploiting the different functional roles played by specific DC subtypes present in tissues. This proposal focuses on the lung, and outlines experiments designed to take several critical steps towards characterizing this system more completely and developing it as a viable treatment for cancer. Our preliminary experiments have shown that there are unique features in both pulmonary DCs. We and others have demonstrated that pulmonary DCs differentially express pattern recognition receptors (such as TLRs), acquire antigen, and present antigen. Our central hypothesis is that both TLR3+CD103+ DC and TLR7+CD11bhi lung DCs, possess the capacity to promote the development of cytotoxic T cells directed to tumor-associated antigens. However, this is only true if the DC is simultaneously 1) presenting the antigen and 2) appropriately stimulated by its corresponding TLR agonist. The proposal addresses a number of critical questions that require resolution in order to fully characterize the system and begin to translate this approach to humans. In Aim 1, we will demonstrate the unique ability of pulmonary DC subtypes to promote cytotoxicity in the presence of either TLR3 or TLR7 ligands. This will fill critical gaps in our knowledge of the mechanisms underlying in vivo DC function and selectivity of TLR expression and activation. In Aim 2, we will investigate the cytokine(s) produced by the activated DC subtype promoting differentiation of cytotoxic T cells. This aim will help define the mechanisms selectively used by endogenous DCs to differentiate T cells. Finally in Aim 3, we will test the anti-tumor properties of pulmonary TLR3+CD103+ DC and TLR7+CD11bhi DC-based treatments in a metastatic lung cancer model. Preliminary data suggest that the specific DC targeting approach may dramatically reduce tumor size in even an aggressive form of cancer. However, more work is needed to fully characterize and develop this model. Overall, achieving these aims will provide substantial advances in understanding the functional roles of lung dendritic cells and explore their potential use in developing endogenous anti-cancer vaccines.